There is a plethora of methods and devices to image biological samples. Predominantly these are variations of confocal microscopy patented by Marvin Minsky 1957. The popularity of confocal microscopy is due to its sectioning capability that enables comparably high resolution in three dimensions and better contrast than can be accomplished with conventional wide-field microscopy. Very often confocal microscopy is combined with fluorescence spectroscopy, where specific particles, e.g. proteins, are marked with fluorophores and thus can be discerned from the background. In most confocal microscopes, the same optics (at least the same objective lens) is used both for the illuminating/exciting and for viewing/detecting the light from the sample under study. An advantage with this configuration is that it mimics the design of the classical wide field microscope and therefore more or less the same optical components can be used. Another advantage is that the illumination and detection are automatically aligned. A further advantage is that by illuminating and viewing with the field of view axis perpendicular to the sample surface the N.A can be large without running into problems such as total reflection.
However, this geometry of conventional confocal microscopy introduces weaknesses and trade-offs that limit the spectrum of applications that can be addressed.
In confocal microscopy, the resolution, i.e. the distance between two distinguishable radiation points, is obtained by limiting the volume (the observation volume) from which emitted light is allowed to reach the detector. Limiting the observation volume is accomplished by combining two approaches: In the dimension orthogonal to the illumination (or equivalent the field of view) axis of the microscope, the extension of observation volume is determined by the focal width in the focal plane of the objective lens. In the dimension along the illumination axis (the field of view axis) of the objective lens, the observation volume is determined by the width of a pinhole placed in the optical conjugate plane in front of the detector. This pinhole is made small enough to block light emanating from points that are outside a certain distance from the focal plane along the direction of the illumination axis. This way, by choosing an objective lens with very small focal width and a small and correctly positioned pinhole, a very high resolution in all three dimensions can be obtained. A further advantage with using the pinhole is that it enhances the contrast since the scattered or fluorescent light emanating from illuminated parts of the sample that are outside the observation volume never reaches the detector.
Nonetheless, in practice this implies a severe trade-off: The pinhole used to filter out the light emanating from outside the focal plane restricts the objective to image more than one point in the focal plane at the time. Thus to obtain an image one has to move (scan) the sample and the observation volume relative to each other. An image synthesized dot by dot and a 1024×1024 frame may take well over a second to accomplish. This complicates the possibility to parallelize the imaging and thus achieve a high throughput.
As mentioned above, the observation volume in conventional confocal microscopy is determined by combining a small focal width with the pin-hole technique. Another way to create an observation volume in three-dimensional space is to use, so called, confocal theta microscopy, Stelzer, E H K, et al. “Fundamental reduction in the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy”. Opt Commun. 111, 536-547, (1994). In this technique the observation volume is instead determined by the use of two objective lenses that are placed such that the axes of their fields of view are orthogonal and that their focal planes intersect. The observation volume is thus limited by the focal width of one detector in one direction and by the focal width of the second detector in an orthogonal second direction. The illumination in this case can be arbitrary as long as it illuminates the observation volume under interest.
From an observation volume perspective theta confocal microscopy removes the need for a pinhole. From a contrast perspective, however, the pinhole is needed to suppress light emanating from outside the observation volume that reaches either of the detectors.
Since confocal microscopes are essentially point-by-point measuring devices, an image of a sample is formed by moving the sample volume, containing whatever sample under study, relative the observation volume, defined by the objective lens of the microscope and the pinhole in front of the detector, and measuring a value of the intensity for each point. The motion continues until an entire two-dimensional (x-y) image is gathered, a process that can be repeated to generate a series of images over time. In addition, the observation volume can also be stepped along the microscope axis to acquire a three-dimensional (x, y, and z) image stack of optical sections. With this vector of values an image in three dimensions can be synthesized and is typically tomographically displayed on a screen.
In early versions of confocal microscopes the sample is placed on a high-precision translation stage that moves the sample systematically in three dimensions until the image vector is generated. In order to create a high frame rate the sample has to be moved along its pre-programmed path at high speed while maintaining sub-micro meter precision. A challenging and expensive task due to the high accelerations of the mass of the sample holder and the stages.
Another approach to attain a high frame rate is laser scanning confocal microscope. Instead of moving the sample, the excitation light beam is expanded and directed to a pair of oscillating galvanometer scanning mirrors that raster-scan the focused beam across the sample volume. The light from the sample is de-scanned through the same mirror set and passed through a conjugate (confocal) pinhole before reaching the detector. The scanning speed is limited by the mechanical specifications of the fastest mirror, which typically scans at a rate of approximately 4 to 5 microseconds per image point (sometimes referred to as a pixel or in three-dimensional space a voxel). Thus, for a 512×512 pixel image collected in a single second, the scanning spot dwells on each pixel for about 4 microseconds. Again, obtaining higher rates is very difficult, if not impossible, due to the fact that the mirror would have to be rapidly accelerated, held at a constant velocity while scanning across the field, then rapidly decelerated and the direction of travel reversed, repeating this cycle for each scanned line.
Another alternative for fast scanning of samples is the (Nipkow) spinning disk method. This confocal microscope is based on a circular, rotating disk that has one or more pinhole arrays that are arranged in a spiral pattern designed to cover the sample volume during one revolution of the disk. Since no accelerations are needed other that rotating the disc, this configuration is mechanically very stable in steady state. Another advantage with this method is that despite the pinholes of the spinning disk several points (pixels) can be viewed in parallel. Combining the high speed and the ability to parallelize very high frame rates have been accomplished by using this technique, exceeding 1000 frames per second. Yet, Spinning disk confocal microscopes are not without their artefacts. One limitation is that light, scattered or emitted, from outside the focal plane can reach the detector by traveling through adjacent pinholes, so called pinhole crosstalk. A second limitation is the low percentage of light (often less than 10%) passing through the pinholes of the disk. The remainder of the light is reflected and may show up as background noise in the detector. Both these side effects limit the signal to noise ratio. A third limitation with the spinning disk method is that the detector typically used to collect the image is either a charge coupled device or a CMOS sensor. Since the integration time of such devices are in the order of milliseconds, the dark current inherent in this type of devices will over said integration time build up noise levels making it virtually impossible to detect single photons.
Gratton et al. (U.S. Pat. No. 7,973,294) proposes to move the sample volume through the observation volume. Instead of having an x-y-z translation stage moving around the sample volume, the sample is placed in a container that is rotating. Thus a very large sample volume can be passed through the observation volume of the confocal microscope with a minimum of acceleration required. When in steady state only the friction of the rotation stage has to be overcome and the container can also be slowly moved in other directions in order to access all parts of the sample volume. However Gratton et al. used a cuvette that contains particles resolved in a liquid. Thus the particles are moving around and it is thus not possible to maintain sub-micrometre precision in this case.
There remains a need for techniques that enable studies, tests, etc., of primarily micro-biologically relevant matter, with a high frame rate (high throughput), high spatial resolution in three dimensions, high sensitivity, over a large area and at an acceptable cost level. None of the state of the art methods are able to meet these requirements simultaneously.